PROJECT SUMMARY Functional plasticity of immune cells is essential to fine-tune their responses to disparate stimuli. Although plasticity may cause unwanted side effects, it may also be harnessed to steer immune responses towards desired outcomes. Innate lymphoid cells (ILC) are lymphocytes devoid of antigen-specific receptors that produce cytokines at early stages of immune responses. Based on specific networks of transcription factors and cytokine profiles, ILC are subdivided into three subsets: T-bet+ ILC1 release IFN-?; GATA3+ ILC2 secrete IL-5 and IL-13; ROR?t+ ILC3 produce IL-22 and IL-17. Recent studies showed that ILC are functionally plastic and heavily influenced by changes in the microenvironment, which raises outstanding questions: 1) While human ILC are functionally plastic in vitro, are they equally flexible in vivo and, if so, what mechanisms are involved? 2) In vivo fate mapping studies in mice have documented ILC plasticity in steady state; what is the extent and impact of ILC plasticity in disease models? 3) What epigenetic circuits control ILC responses to fluctuations in the microenviroment? We will address these questions in three aims. Aim 1 presents the first demonstration of transitional ILC subsets in human tonsils with features of both ILC3 and ILC1, which is evidence for ILC3?ILC1 conversion in vivo. We also present data indicating that the IKZF3-encoded transcription factor Aiolos is required for transition. We will test the hypothesis that Aiolos cooperates with other transcription factors to drive human ILC3?ILC1 conversion, using in vitro and in vivo approaches. We will perform chromatin studies of ILC3?ILC1 conversion to further define regulatory circuits and transcription factors that govern ILC3/ILC1 plasticity. Clonal analyses of transitional ILC subsets will be employed to corroborate their homogeneity and developmental trajectory. In Aim 2, we propose to precisely characterize ILC3/ILC1 transitional populations in the human intestine by mass cytometry and scRNAseq, as they are not as clearly defined as those in tonsils. Moreover, we propose in vivo mouse studies to determine whether diseases that alter intestinal microenvironment induce ILC3/1 plasticity and, in turn, whether plasticity impacts immune responses during gastrointestinal infections and IBD. These experiments will be carried out in Ror?t-reporter mice and in ILC3-deficient mice reconstituted with either ILC3 or ex-ILC3. In Aim 3, we will test the hypothesis that in diseases that induce type 1 and type 3 polarizing cytokines, ILC2 in the gut and skin convert into ILC1/3. We will track ILC2 plasticity in vivo using reporter mice and test the impact of plastic ILC2 in models of infections, IBD and skin inflammation in adoptive transfer experiments. We will also define the regulatory elements controlling ILC2 plasticity in chromatin studies. We hope that our expertise and leadership in the ILC field will yield an integrated view of ILC functional adaptation in immunity.